Structure and Function of Glycosyltransferases: To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown following: (I) Glycosyltransferases have flexible loop(s) in the vicinity of their catalytic pocket which undergo conformational changes upon donor substrate binding and create the acceptor binding site: (II) In the metal-ion dependent enzymes, the metal ion binding site is generally at the amino terminal hinge region of the flexible loop.(III) Glycosyltransferases interact with the add-on domains: To diversify the catalytic activity towards less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of glycosyltransferases either interact (1) with an additional protein, or have acquired add-on domains at the C-terminus or acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme, the b4Gal-T1, after conformational changes in the flexible loops to a closed conformation, interacts with a mammary gland-specific protein, alpha-lactalbumin (LA) at its carboxyl terminal end, changing the acceptor specificity of the enzyme towards less preferred acceptor glucose. LA protein, although not linked to b4Gal-T1, acts as an add-on domain. Several other glycosyltransferases have been shown or suggested to require an activating protein. In contrast to two interacting proteins, the catalytic domains of polypeptide a-N-Acetylgalactosaminyl-transferases (ppGalNAc-Ts) have a lectin domain that is linked to the C-terminus of the catalytic domain via a linker region and determines the specificity towards a peptide or a glycopeptide. The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, while the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize O-alpha-GalNAc moiety on the glycopeptide. Also in this category is the alpha-1,6-Fucosyltransferase (FUT8), where an SH3 domain has been identified that is linked at the C-terminus of the catalytic domain. (IV) To understand the oligosaccharide acceptor specificity of b1,4-galactosyltransferase-I (b4Gal-T1) enzyme, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates bind to the crevice present at the acceptor substrate binding site of the b4Gal-T1 molecule. Now we have seven crystal structures of b4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a b1-6 glycosidic link and that are analogous to either N-glycan or i/I- antigen. In the crystal structure of the complex of b4Gal-T1 with I-antigen analogue pentasaccharide, the b1-6 branched GlcNAc moiety is bound to the sugar acceptor binding site of the b4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetra-saccharide moiety does not interact with the previously found extended sugar binding site on the b4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Y276, Trp310, and Phe356. Results from the present and previous studies suggest that b4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate. (V) A few residues in the catalytic pocket determine the donor sugar specificity of glycosyltransferases: Role of a single amino acid in the evolutionary divergence of invertebrate and vertebrate glycoconjugates: (a) Mutations in catalytic pocket of b4Gal-T1 change its donor specificity: Based on the structural information, we have previously shown, that the residue Tyr/Phe289 in the catalytic pocket of b4Gal-T1, which is conserved among all vertebrate homologs, when mutated to Leu or Ile broadens the donor substrate specificity of the enzyme to 2substituants of galactose i.e., GalNAc or 2-keto-galactose or 2-azido-galactose. In invertebrates in the b4Gal-T homologs there is an Ile residue at the corresponding position of Tyr and they are b4GalNAc-T enzymes. Mutation of the Ile residue to Tyr in Drosophila b4GalNAc-T1 converts the enzyme to a b4Gal-T1 by reducing its N-acetyl-galactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold.(b) Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) broadens the donor specificity: We have mutated bovine a1,3-galactosyltransferse (a3Gal-T) enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives by mutating the sugar donor-binding residues at His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant a3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants 280SGG282 and 280AGG282 with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of glycoprotein, thus enabling the detection of LacNAc moiety by a chemiluminescence method. This makes it possible to use these mutants, (1) for the detection of alterations in the glycosylation patterns in many pathological states, such as cancers and rheumatoid arthritis, and (2) in the glycoconjugation and assembly of nano-particles for the targeted drug delivery of bioactive-agents. (VI) The N-acetyl group of the donor sugar is generally embedded in a hydrophobic pocket of the enzyme. In both mutant enzymes,Y289L-b4Gal-T1 and SGG-a3Gal-T, the N-acetyl moiety of the donor sugar GalNAc, is embedded in a hydrophobic pocket that allows the substitution of this moiety by CH2-CO-CH3 group. We have now shown that the N-acetyl groups of the donor sugars GlcNAc and GalNAc of the N-acetylglucosaminyl- and N-acetylgalactosaminyl-transferases are generally embedded in a cavity or a hydrophobic pocket which can also accommodate a ketone group in the N-acetyl-binding pocket, making it possible to attach to the chemical handle affinity probes for detection, isolation, and characterization of the product and linking biomolecules. (VII)Galectin-1 as a fusion partner has been used for the production of soluble and folded human beta-1, 4- Galactosyltransferase-T7 in E. coli. (VIII)Crystal structures of the catalytic domain of Drosophila and human beta-1,4-galactosyltransferase-T7: Gene knockout studies in Drosophila have shown that that the b4Gal-T7, one member of the b4Gal-T family that transfers Gal to Xylose on proteoglycans, is essential for species survival while lack of b4Gal-T1 gene led to multiple disorders. However, mutations in the human b4Gal-T7 are known to cause skin fibroblasts of an Ehlers-Danlos syndrome. The catalytic domain of human b4Gal-T7 exhibits a 39% amino acid sequence similarity with the catalytic domain of human b4Gal-T1, while it shows a 68% sequence similarity with the catalytic domain of b4Gal-T7 from Drosophila. Having established the crystal structure of b4Gal-T7 from Drosophila, we have now in the FY 11-12 extended our crystal structure studies on human b4Gal-T7. We solved crystal structure of human b4Gal-T7, at 2.7 A resolution, by molecular replacement using Drosophila b4GalT7 crystal structure as a model structure. The structure has been refined and shows longer flexible loop and reveals unexpected features that were not observed in the Drosophila b4GalT7 structure and are currently being investigated.